HUMAN IMMUNODEFICIENCY VIRUS
TYPES 1 and 2
(Recombinant and Synthetic Peptides)
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GS HIV-1/HIV-2 PLUS O EIA
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Recombinant and Synthetic Peptide Enzyme
Immunoassay (EIA) for the Detection of Antibody to
Human Immunodeficiency Virus Types 1
(Groups M and O) and/or 2 (HIV-1/HIV-2) in Human
Serum, Plasma, or Cadaveric Serum Specimens
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For In Vitro Diagnostic Use
32588 • 480 Tests
32589 • 960 Tests
25256 • 4800 Tests
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
LEXICON
For In Vitro Diagnostic Use
European Conformity
Catalog Number
Consult Instructions for Use
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Temperature Limit
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Number of Tests
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Manufactured by
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Authorized Representative in the
European Community
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Wash Solution Concentrate (30X)
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Chromogen: TMB Solution
Substrate Buffer
Stopping Solution
Biohazard
WARNING
Corrosive
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
CONTENTS
1 - NAME AND INTENDED USE
2 - SUMMARY AND EXPLANATION OF THE TEST
3 - BIOLOGICAL PRINCIPLES OF THE PROCEDURE
4 - REAGENTS
5 - WARNINGS FOR USERS
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6 - PRECAUTIONS FOR USERS
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7 - REAGENT PREPARATION AND STORAGE
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8 - SPECIMEN COLLECTION, PREPARATION, AND
STORAGE
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9 - GS HIV-1/HIV-2 PLUS O EIA PROCEDURE
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10- QUALITY CONTROL - VALIDATION OF RESULTS
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11- INTERPRETATION OF RESULTS
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12- LIMITATIONS OF THE PROCEDURE
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13- PERFORMANCE CHARACTERISTICS OF SERUM AND
PLASMA TESTING
14- PERFORMANCE CHARACTERISTICS OF CADAVERIC
SPECIMEN TESTING
15- REFERENCES
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
1 - NAME AND INTENDED USE
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The GS HIV-1/HIV-2 PLUS O EIA is an enzyme immunoassay
utilizing recombinant proteins and synthetic peptides for the
detection of antibodies to HIV-1 (Groups M and O) and/or HIV-2
in human serum and plasma. It is indicated as a screening test
for specimens from individual human donors, including donors
of whole blood, blood components, and source plasma, and
from other living donors. It is also intended for use in testing
plasma and serum specimens to screen organ donors when
specimens are obtained while the donor’s heart is still beating,
and in testing blood specimens from cadaveric (nonheartbeating) donors. In addition, it is intended as an aid in the
diagnosis of infection with HIV-1 and/or HIV-2. The assay is not
intended for use on cord blood specimens.
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The GS HIV-1/HIV-2 PLUS O EIA is intended for manual use and
also for use with the ORTHO® Summit System (OSS) in the
screening of blood donors.
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The GS HIV-1/HIV-2 PLUS O EIA is intended for manual use and
also for use with the EVOLIS® and Elite™ Automated Microplate
Systems as an aid in the diagnosis of infection with HIV-1 and/or
HIV-2.
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2 - SUMMARY AND EXPLANATION OF THE TEST
The acquired immunodeficiency syndrome (AIDS) is caused by
viruses transmitted by sexual contact, exposure to blood
(including sharing contaminated needles and syringes) or certain
blood products, or transmitted from an infected mother to her
fetus or child during the perinatal period.1 Additionally,
transmission of these viruses can occur through tissue
transplantation.2 Human Immunodeficiency Virus Type 1 (HIV-1)
has been isolated from patients with AIDS and AIDS-related
complex (ARC).3-5 HIV-1 was thought to be the sole causative
agent of these syndromes until 1986, when a second type of
Human Immunodeficiency Virus (Human Immunodeficiency Virus
Type 2 or HIV-2) was isolated and also reported to cause
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
AIDS.6-7 Since the initial discovery, hundreds of cases of HIV-2
infection have been documented worldwide, including cases of
AIDS related to HIV-2.8 In the United States, there have been
more than 80 cases of infection with HIV-2 reported, including
three potential blood donors.9-14
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This second immunodeficiency virus is similar to, but distinct
from, HIV-1. Both viruses have similar morphology and
lymphotropism,15 and the modes of transmission appear to be
identical.8,16 The HIV-1 and HIV-2 genomes exhibit about 60%
homology in conserved genes such as gag and pol, and 39-45%
homology in the envelope genes.17 Serologic studies have also
shown that the core proteins of HIV-1 and HIV-2 display frequent
cross-reactivity, whereas the envelope proteins are more
type-specific.18
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Within the two major HIV types, there is significant variation as
well. By analyzing sequences of representative strains, HIV-1
has been divided into three groups: Group M (for major),
including at least ten subtypes (A through J); Group O (for
outlier); and Group N (for non-M, non-O).19-21 Similarly, the HIV-2
strains have been classified into at least five subtypes (A through
E).22 Some HIV-1 variants share ≤ 50% homology in their
envelope genes with the sequences of more common prototype
strains.
Despite some degree of immunological cross-reactivity between
types and subtypes of HIV, reliable detection of the more
divergent strains may only be achieved by incorporating specific
sequences into the assay design. In one study, detection of
HIV-2 positive samples by licensed HIV-1 antibody kits ranged
from 60% to 91%, depending on the test used.23 Detection of
HIV-1 Group O samples by HIV-1 and HIV-1/HIV-2 assays varied
from 0% to 100% in studies with U.S.-licensed and European
test kits.24,25 The GS HIV-1/HIV-2 PLUS O EIA incorporates
highly conserved recombinant and synthetic peptide sequences
representing HIV-1 (Groups M and O) and HIV-2.26-32 It was
developed to improve sensitivity and specificity of detection of
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
antibodies to HIV-1 and/or HIV-2 for blood and plasma
screening, and as an aid in the diagnosis of HIV infection.
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Any specimen that reacts in an initial test (is initially reactive) with
the GS HIV-1/HIV-2 PLUS O EIA must be retested in duplicate
with the GS HIV-1/HIV-2 PLUS O EIA. Initially reactive specimens
that are reactive in either one or both duplicates from the repeat
testing are referred to as repeatedly reactive. Repeatedly
reactive specimens may contain antibodies to either HIV-1 or
HIV-2. Therefore, additional, more specific or supplemental tests
for antibodies to both HIV-1 and HIV-2, such as Western blot or
immunofluorescence, must be performed to verify the presence
of antibodies to HIV-1 or HIV-2. Recommendations for
appropriate use of such additional tests may be issued
periodically by the United States Public Health Service.
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3 - BIOLOGICAL PRINCIPLES OF THE PROCEDURE
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The GS HIV-1/HIV-2 PLUS O EIA is an enzyme immunoassay
based on the principle of the direct antibody sandwich
technique. Microwell strip plates (the solid phase) are coated
with purified antigens: gp160 and p24 recombinant proteins
derived from HIV-1; a peptide representing the immunodominant
region of the HIV-2 transmembrane glycoprotein, gp36; and a
synthetic polypeptide mimicking an artificial (i.e., encoded by no
existing virus) HIV-1 Group O specific epitope.
Samples and controls are added to the wells along with
Specimen Diluent. The Specimen Diluent contains a dye that
changes color from purple to blue when combined with a sample
or control. The wells are incubated and then washed. The next
step is the addition of a colored Conjugate Solution (green),
which contains peroxidase-conjugated antigens (peptides
mimicking various immunodominant epitopes of the HIV-1 and
HIV-2 transmembrane glycoproteins, and a p24 recombinant
protein). The wells are then incubated. If HIV-1 and/or HIV-2
antibody is present, it will bind to the antigen coated on the well
and to the peroxidase-conjugated antigens in the Conjugate.
The antigen-antibody-antigen complexes remain bound to the
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
well during the subsequent wash step, which will remove any
unbound materials. Working TMB Solution is added to the plate
and allowed to incubate. A blue or blue-green color develops in
proportion to the amount of HIV antibody present in the sample.
Color development is stopped by the addition of acid, which
changes the blue-green color to yellow. The optical absorbance
of specimens and controls is determined spectrophotometrically
at a wavelength of 450 nm.
4 - REAGENTS
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GS HIV-1/HIV-2 PLUS O EIA Product Description
Product No. 32588 (480 Tests), 32589 (960 Tests), 25256 (4800 Tests)
Contents
Microwell plate with adsorbed purified HIV-1
and HIV-2 antigens.
ProClin 150 preservative
Tabs are labeled “BB”
Sodium chloride
Tween 20™
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R2 • Wash Solution
Concentrate (30X)
2, 3, or ** bottles
(120 mL)
R3 • Specimen Diluent
1, 1, or 5 bottle(s)
(100 mL)
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Component
R1 • HIV-1/HIV-2 PLUS O
Microwell Strip Plate
5, 10, or 50
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• Bovine proteins
• Buffer with protein stabilizers
• 0.1% ProClin 300 preservative
• Sample indicator dye
• Human serum; negative for HIV and HCV
antibodies and HBsAg
• 0.005% Gentamicin sulfate
• 0.16% ProClin 950 preservative
• Human HIV-1 antibody in human
serum/plasma
• Non-reactive for HBsAg and antibodies to
HCV
• 0.005% Gentamicin sulfate
• 0.16% ProClin 950 preservative
• Human HIV-2 antibody in human
serum/plasma
• Non-reactive for HBsAg and antibodies to
HCV
• 0.005% Gentamicin sulfate
• 0.16% ProClin 950 preservative
• Rabbit HIV-1 Group O antibody in human
serum, negative for HIV and HCV antibodies
and HBsAg
• 0.005% Gentamicin sulfate
• 0.16% ProClin 950 preservative
Dilute in Specimen Diluent
as described.
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C0 • Negative Control
1, 1, or 5 vial(s)
(8 mL)
Preparation
Use as supplied. Return
unused strips/plates to
pouch and reseal. Do not
remove desiccant.
Dilute 1:30 with deionized
water. Clinical laboratory
reagent water (CLRW) is
acceptable.
Use as supplied.
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C1 • HIV-1 Positive Control
1, 2, or 10 vial(s)
(1.4 mL)
C2 • HIV-2 Positive Control
1, 2, or 10 vial(s)
(1.4 mL)
C3 • HIV-1 Group O Positive
Control
1, 2, or 10 vial(s)
(1.4 mL)
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Dilute in Specimen Diluent
as described.
Dilute in Specimen Diluent
as described.
Dilute in Specimen Diluent
as described.
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
GS HIV-1/HIV-2 PLUS O EIA Product Description(cont.)
Component
Contents
•
•
•
•
•
•
•
•
•
•
•
1N Sulfuric acid (H2SO4)
•
Dilute in Conjugate Diluent
as described.
Use as supplied.
Use as supplied.
Dilute with Substrate
Buffer as described.
Use as supplied.
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R5 • Conjugate Diluent
1, 1, or 5 bottle(s), (120 mL)
R8 • Substrate Buffer
1, 1, or 5 bottle(s)
(120 mL)
R9 • Chromogen (11X)
1, 1, or 5 bottles(s) (12 mL)
R10 • Stopping Solution
1, 1, or ** bottle(s), (120 mL)
Preparation
Purified HIV-1 and HIV-2 antigens labeled with
peroxidase
Buffer with protein stabilizers (bovine and
caprine)
0.005% Gentamicin sulfate
0.5% ProClin 300 preservative
Green dye
Buffer with protein stabilizers (bovine)
0.1% ProClin 300 preservative
Hydrogen peroxide
Citric acid/Sodium acetate buffer
DMSO
Tetramethylbenzidine (TMB)*
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R4 • Conjugate Concentrate
(11X)
1, 1, or 5 vial(s), (12 mL)
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* Note: Tetramethylbenzidine is a non-carcinogenic and non-mutagenic chromogen for peroxidase.33,34
** Wash Solution Concentrate and Stopping Solution must be purchased separately for the 50 plate (4800 test)
kit. Refer to catalog number 25261 for the Wash Solution Concentrate and catalog number 25260 for the
Stopping Solution. These reagents are included in the 5 plate (480 test) and 10 plate (960 test) kits.
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Store kit at 2-8°C. Bring all reagents except Conjugate
Concentrate to room temperature (18-30°C) before use. Return
reagents to 2-8°C after use. Return unused strips/plates to
pouch and reseal. Do not remove desiccant. Store strips/plates
at 2-8°C.
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5 - WARNINGS FOR USERS
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For In Vitro Diagnostic Use
WARNING: FDA has licensed this test for use with serum,
plasma, and cadaveric serum specimens only. Use of this
licensed test kit with specimens other than those specifically
approved for use with this test kit may cause inaccurate test
results.
1. This test kit should be handled only by qualified personnel
trained in laboratory procedures and familiar with their
potential hazards. Handle appropriately with the requisite
Good Laboratory Practices. Wear appropriate protective
clothing, including lab coat, eye/face protection, and
disposable gloves (synthetic, non-latex gloves are
recommended) while handling kit reagents and clinical
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
specimens. Wash hands thoroughly after performing the
test.
2. Do not smoke, drink, or eat in areas where specimens or kit
reagents are being handled.
3. Do not pipette by mouth.
4. The following is a list of potential chemical hazards
contained in some reagents (refer to Product Description
chart):
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a. 0.005% Gentamicin Sulfate, a biocidal preservative, which
is a known reproductive toxin, photosensitizer, and
sensitizer; prolonged or repeated exposure may cause
allergic reaction in certain sensitive individuals.
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b. WARNING: Components R3, R4 and R5 contain 0.1% or
0.5% ProClin 300.
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H317: May cause allergic skin
reaction.
P280: Wear protective
gloves/protective clothing/eye
protection/face protection.
P302 + P352: IF ON SKIN: Wash with plenty of
soap and water.
P333 + P313: If skin irritation or rash occurs:
Get medical advice/attention.
P501: Dispose of contents and
container in accordance with
local, regional, national, and
international regulations.
ProClin 300 (0.1% or 0.5%) is a biocidal preservative that
is irritating to eyes and skin, may be detrimental if enough
is ingested, and may cause sensitization by skin contact;
prolonged or repeated exposure may cause allergic
reaction in certain sensitive individuals.
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
c. DANGER! The Stopping Solution (R10) contains 1N
Sulfuric Acid.
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H314: Causes severe skin burns and
eye damage.
H290: May be corrosive to metals.
P280: Wear protective
gloves/protective clothing/eye
protection/face protection.
P301 + P330 IF SWALLOWED: Rinse mouth.
+ P331: Do NOT induce vomiting.
P305 + P351 IF IN EYES: Rinse cautiously
+ P338: with water for several minutes.
Remove contact lenses, if
present and easy to do.
Continue rinsing.
P501: Dispose of contents and
container in accordance with
local, regional, national, and
international regulations.
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The 1N Sulfuric Acid (H2SO4) Stopping Solution is
severely irritating or corrosive to eyes and skin,
depending on the amount and length of exposure; greater
exposures can cause eye damage, including permanent
impairment of vision. In case of contact with eyes, rinse
immediately with plenty of water and seek medical
advice. Keep away from strong bases, reducing agents,
and metals; do not pour water into this component.
Waste from this material is considered hazardous acidic
waste, however if permitted by local, regional, and
national regulations, it might be neutralized to pH 6-8 for
non-hazardous disposal.
5. The GS HIV-1/HIV-2 PLUS O EIA contains human blood
components. No known test method can offer complete
assurance that infectious agents are absent. Therefore, all
human blood derivatives, reagents, and human specimens
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
should be handled as if capable of transmitting infectious
disease, following recommended Standard and Universal
Precautions for bloodborne pathogens as defined by OSHA,
Biosafety Level 2 guidelines from the current CDC/NIH
Biosafety in Microbiological and Biomedical Laboratories35,
WHO Laboratory Biosafety Manual36, and/or local, regional,
and national regulations. The following human blood
derivatives are found in this kit:
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a. Human source material used in the preparation of the
Negative Control (C0) and as a diluent for the Positive
Controls (C1, C2, and C3) has been tested and found
non-reactive for Hepatitis B surface antigen (HBsAg), and
antibodies to Hepatitis C virus (HCV Ab) and human
immunodeficiency virus (HIV-1 and HIV-2).
b. Human source material, containing HIV-1 and HIV-2
human antibody used in the preparation of the Positive
Controls (C1 and C2) has been heat-treated. It has been
tested and found nonreactive for Hepatitis B surface
antigen (HBsAg) and antibodies to Hepatitis C virus (HCV
Ab).
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6. Biological spills: Human source material spills should be
treated as potentially infectious.
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Spills not containing acid should be immediately
decontaminated, including the spill area, materials, and any
contaminated surfaces or equipment, with an appropriate
chemical disinfectant that is effective for the potential
biohazards relative to the samples involved (commonly a
1:10 dilution of household bleach, 70-80% Ethanol or
Isopropanol, an iodophor [such as 0.5% Wescodyne Plus,
EPA Registration #4959-16-52], or a phenolic), and wiped
dry.37-40
Spills containing acid should be appropriately absorbed
(wiped up) or neutralized, and then the area wiped with one
of the chemical disinfectants. Material used to absorb the
spill may require biohazardous waste disposal.
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
NOTE: DO NOT PLACE SOLUTIONS CONTAINING BLEACH
INTO THE AUTOCLAVE.
7. Dispose of all specimens and material used to perform the
test as though they contain an infectious agent. Laboratory,
chemical, or biohazardous wastes must be handled and
discarded in accordance with all local, regional, and national
regulations.
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8. There are no health hazards associated with the intact
desiccant packet; do not cut, split, or otherwise compromise
it as dusts that may be generated could pose a health
hazard. If the desiccant has been compromised, do not
remove it from the plate pouch.
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9. Complete hazard information and precautions are located in
the Safety Data Sheet (SDS) available at bio-rad.com or
upon request from Bio-Rad Technical Services.
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6 - PRECAUTIONS FOR USERS
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1. Do not use any kit components beyond their stated
expiration date.
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2. The reagents that may be used with different lots of the
GS HIV-1/HIV-2 PLUS O EIA kit are the Chromogen (R9),
Substrate Buffer (R8), Wash Solution Concentrate (R2), and
Stopping Solution (R10). Do not mix any other reagents from
different lots. Any lot number of the following reagents may
be used with this assay provided they have the correct
catalog number and are not used beyond their labeled
expiration date:
•
Chromogen (R9) - Catalog #26182
•
Substrate Buffer (R8) - Catalog #26181
•
Wash Solution Concentrate (R2) - Catalog #25261
•
Stopping Solution (R10) - Catalog #25260
3. The tabs at the end of the microwell strips are labeled with
product code “BB.” Do not use strips that have other
product codes with this kit.
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
4. Exercise care when opening vials and removing aliquots to
avoid microbial contamination of the reagents.
5. Use a clean, disposable container for the Conjugate
Solution. Exposure of the Conjugate to sodium azide will
result in its inactivation.
6. Avoid exposing Chromogen or the Working TMB Solution to
strong light during storage or incubation. Do not allow the
chromogen solutions to come into contact with an oxidizing
agent.
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7. Use clean, polypropylene containers (do not use
polystyrene containers) to prepare and store the Working
TMB Solution. If glassware must be used, pre-rinse
thoroughly with 1N sulfuric or hydrochloric acid followed by
at least three washes of deionized water. Be sure that no
acid residue remains on the glassware. If polypropylene
containers are to be reused, they should be cleaned in
accordance with a cleaning process validated by the testing
facility.
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8. Bring all reagents except Conjugate Concentrate to room
temperature before use.
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9. Clinical samples may contain very high levels of HIV
antibody. Therefore, care must be exercised when
dispensing samples to avoid cross contamination through
aerosols or carryover. For manual pipetting of controls and
specimens, use an individual pipette tip for each sample and
do not allow other parts of the pipetting device to touch the
rim or interior of the specimen container. Consider using new
stoppers/caps to seal specimen tubes after use, to avoid
errors or contamination of the work area while recapping
tubes.
10. Handle the Negative Control and Positive Controls in the
same manner as patient specimens.
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
11. If a specimen or reagent is inadvertently not added to a well,
the assay results will read negative. Reagents of this kit have
been color-coded to enable confirmation of the addition of
specimens/controls and Working Conjugate Solution.
12. Inadequate adherence to package insert instructions may
cause erroneous results.
13. Use only adequately calibrated equipment with this assay.
14. Use of dedicated equipment is recommended if equipment
performance validations have not precluded the possibility
of cross-contamination.
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15. The GS HIV-1/HIV-2 PLUS O EIA performance is highly
dependent upon incubation times and temperatures and
effective washing. Temperatures outside of the validated
ranges may result in invalid assays. Incubation temperatures
should be carefully monitored using calibrated
thermometers, or equivalent.
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16. Caution: Certain washer conditions, such as partially
blocked cannulae that are not detected by the OSP
instrument, can lead to sub-optimal washing and false
reactive test results. It is recommended that users of
ORTHO® Summit System carefully verify that the washing
system is clear and operating properly before performing
an assay.
7 - REAGENT PREPARATION AND STORAGE
Working Conjugate Solution
Note: 1:11 dilution. Bring Conjugate Diluent (R5) to room
temperature. Use only the matched lot of Conjugate
Concentrate provided with the kit master lot being used.
(See PRECAUTIONS FOR USERS section, item 2, page 12.)
Invert Diluent (colorless to pale straw) and Conjugate
Concentrate (R4, green) to mix before using. Prepare a 1:11
dilution for each strip to be tested by adding 100 μL of
Conjugate Concentrate to 1 mL of Conjugate Diluent in a clean,
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
plastic tube. Use the following table as a guide. Ensure that the
volume of Working Conjugate Solution that is prepared will be
adequate for the entire run. Mix well. Working Conjugate
Solution should be green. Note Concentrate lot number, date
and time of preparation, and date and time of expiration of the
Working Conjugate Solution. Working Conjugate Solution is
stable for 8 hours at room temperature. Mix working solution
prior to use.
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Return Conjugate Concentrate to the refrigerator immediately
after use. To avoid contamination of Conjugate, wear clean
gloves and do not touch tips of pipettes.
Number of Strips
to be used
2
3
4
5
6
100
200
300
400
500
600
Amount of Conjugate
Diluent (mL)
1
2
3
4
5
7
8
9
10
11
12*
700
800
900
1000
1100
1200
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8
9
10
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Amount of Conjugate
Concentrate (μL)
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Preparation of Working Conjugate Solution by Strip
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* Complete Plate
Preparation of Working Conjugate Solution by Plate
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Number of Complete
Plates to be used
1
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1.2
12
2
3
4
5
6
7
8
9
10
2.4
3.6
4.8
6.0
7.2
8.4
9.6
10.8
12.0
24
36
48
60
72
84
96
108
120
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Amount of Conjugate
Diluent (mL)
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Amount of Conjugate
Concentrate (mL)
Working TMB Solution
Note: 1:11 dilution. Bring Chromogen and Substrate Buffer to
room temperature. Invert the Chromogen and Substrate Buffer
to mix before using. Prepare a 1:11 dilution for each strip to be
tested by mixing 100 μL of Chromogen to 1 mL of Substrate
Buffer in a clean, polypropylene container (do not use a
polystyrene container). Note Chromogen lot number, date and
time of preparation, and date and time of expiration (8 hours
from preparation) on container. Mix Working Solution gently
when combined and again just prior to use. Working TMB
Solution should be kept in the dark at room temperature and
used within 8 hours.
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FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Chromogen should be colorless to slightly yellow. Any other
color indicates that the reagent is contaminated. Do not use this
reagent. The Working TMB Solution should be colorless. A
distinct blue color indicates that the reagent is contaminated.
Discard the Working TMB Solution and prepare fresh reagent in
a clean container.
Prepare only the amount of the reagent to be used within 8
hours, ensuring that the volume of diluted reagent will be
adequate for the entire run. Extra Chromogen is provided. Use
the following table as a guide:
1
2
3
4
5
6
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Amount of
Chromogen (μL)
100
200
300
400
500
600
700
1
2
3
4
5
6
7
9
10
11
12*
800
900
1000
1100
1200
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9
10
11
12
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* Complete Plate
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Amount of Substrate
Buffer (mL)
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Number of Strips
to be used
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Preparation of Working TMB Solution by Strip
Number of Complete Plates
to be used
3
4
5
6
7
8
9
10
1.2
2.4
3.6
4.8
6.0
7.2
8.4
9.6
10.8
12.0
12
24
36
48
60
72
84
96
108
120
R
R
EF
Amount of
Chromogen (mL)
Amount of Substrate
Buffer (mL)
2
ER
1
EN
Preparation of Working TMB Solution by Plate
FO
Wash Solution
Prepare Wash Solution by adding one part Wash Solution
Concentrate (30X) to 29 parts of water (e.g., 120 mL of Wash
Solution Concentrate to 3480 mL of water). Any lot of Wash
Solution Concentrate, provided it is catalog number 25261 and
within its labeled shelf life, may be used with this assay. Use
deionized or distilled water. Clinical laboratory reagent water is
acceptable. The diluted Wash Solution can be stored at room
temperature for up to four weeks in a plastic container. Note the
lot number, date prepared, and expiration date on the container.
Prepare a sufficient quantity of Wash Solution to complete a full
run.
16
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
8 - SPECIMEN COLLECTION, PREPARATION, AND
STORAGE
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Serum, plasma, or cadaveric serum specimens may be used in
the test. The following anticoagulants, including those in both
glass and plastic tubes, have all been evaluated and found to be
acceptable: EDTA, sodium and lithium heparin, sodium citrate,
CPD, CPDA-1, and ACD. Samples that are collected into
anticoagulant tubes should be filled as labeling indicates to
avoid improper dilution. SST tubes are acceptable for use, both
with and without activator. Specimens with observable
particulate matter should be clarified by centrifugation prior to
testing. No clinically significant effect has been detected in assay
results of serum or plasma samples with increased levels of
protein, lipids, bilirubin, or hemolysis, or after heat inactivation of
patient samples. Cadaveric serum samples with increased levels
of hemolysis have been tested, and no clinically significant effect
has been detected in assay results. Note: Cadaveric serum
samples with increased levels of protein, lipids, bilirubin, or
microbiological contaminants have not been available to
evaluate with this assay.
FO
R
R
EF
Specimens may be stored at 2-8°C for 7 days. For long-term
storage, the specimens should be frozen (at -20°C or colder).
Samples should not be used if they have incurred more than 5
freeze-thaw cycles. Mix samples thoroughly after thawing.
Note: If specimens are to be shipped, they should be packed
in compliance with Federal Regulations covering the
transportation of etiologic agents. Studies have demonstrated
that specimens may be shipped refrigerated (2-8°C) or at
ambient temperature (≤ 37°C) for up to 7 days. For shipments
that are in transit for more than 7 days, specimens should be
kept frozen (-20°C or lower).
This kit is not licensed for use with specimens other than serum,
plasma, or cadaveric serum specimens. This kit is not intended
for use on saliva/oral fluid or urine samples.
17
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
9 - GS HIV-1/HIV-2 PLUS O EIA PROCEDURE
Materials Provided
See REAGENTS section on page 7.
Materials Required But Not Provided
1. Precision pipettes that deliver 20 to 200 μL, 1 mL, 10 mL,
25 mL, and 50 mL, as needed (accurate within ±10%), and
corresponding pipette tips; multichannel pipettors capable
of delivering 25 μL and 100 μL are optional.
2. Appropriately sized graduated cylinders.
LY
3. Dry-heat incubator capable of maintaining 37 ± 2°C.
SE
O
N
4. Microwell plate or strip washer qualified for use with this
assay. The washer must be capable of dispensing 400 μL
per well, cycling 5 times, and soaking for 30-60 seconds
between each wash.
EN
C
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5. Microwell strip reader qualified for use with this assay. The
spectrophotometer should have the following specifications
at wavelength 450 nm:
R
EF
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Bandwidth: 10 nm HBW (Half Band Width) or equivalent
Absorbance Range: 0 to 2 AU (Absorbance Units)
Repeatability: ± (0.5% + 0.005) AU
Linearity or Accuracy: 1% from 0 to 2.0 AU
FO
R
The instrument should contain a reference filter for reading
at 615 to 630 nm.
6. The GS HIV-1/HIV-2 PLUS O EIA may be used with the
EVOLIS® and Elite™ instrument systems as an aid in the
diagnosis of infection with HIV-1 and/or HIV-2. Note: For
availability of these systems in your area, contact Bio-Rad
Technical Service.
7. The GS HIV-1/HIV-2 PLUS O EIA is approved for use with
the ORTHO® Summit System (OSS) as a screening test for
human serum and plasma specimens. Within the OSS the
operator can process by three different modes: manually
(manual pipetting + manual processing), semi-automated
(automated pipetting + manual processing), or automated
18
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
(automated pipetting + automated processing). The
automated mode can consist of the stand-alone ORTHO
VERSEIA® Pipetter with the stand-alone ORTHO® Summit
Processor (OSP), or the ORTHO VERSEIA® Pipetter
integrated with the OSP as the ORTHO VERSEIA® Integrated
Processor (VIP). The GS HIV-1/HIV-2 PLUS O EIA ORTHO®
Assay Protocol Disk is available from ORTHO® Clinical
Diagnostics.
LY
8. Household bleach (5% to 8% sodium hypochlorite) which
may be diluted to a minimum concentration of 10% bleach
(or 0.5% sodium hypochlorite). Alternative disinfectants
include 70% ethanol or 0.5% Wescodyne™.
O
N
9. Paper towels or absorbent pads for blotting.
SE
10. Labeled null strips for testing partial plates.
EN
C
E
U
11. Clean polypropylene container for preparation of Working
TMB Solution (do not use polystyrene). Clean container for
preparation of Working Conjugate Solution, 15 or 50 mL.
ER
12. Deionized or distilled water. Clinical laboratory reagent water
is acceptable.41
EF
13. Gloves.
R
14. Laboratory timer.
FO
R
15. EIA reagent reservoirs (optional).
16. Plate Sealers (Catalog #0210-00, or equivalent) are required,
except on an approved microplate processor.
Preliminary Statements
1. The expected run time for this procedure is approximately
2.5 - 3 hours from initiation of the first incubation step. Each
run of this assay must proceed to completion without
interruption after it has been started. The maximum
allowable time from start of pipetting to start of incubation is
≤ 1 hour.
2. Using the kit controls, three wells of Negative Control, one
well of HIV-1 Positive Control, one well of HIV-2 Positive
19
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Control, and one well of HIV-1 Group O Positive Control
must be run on each plate. Assay validity and the cutoff for
patient samples are determined by the controls on each
individual plate.
3. Do not splash controls, specimens, or reagents between
microwells of the plate.
4. Cover plates for each incubation step using plate sealers or
other appropriate means to minimize evaporation.
LY
5. Avoid exposure of the plates to light during the final
incubation step (following the addition of Working TMB
Solution).
SE
O
N
6. Adhere to the recommended time constraints for the use of
the Working TMB Solution (8 hours), Working Conjugate
Solution (8 hours), and Working Wash Solution (4 weeks).
U
7. Avoid the formation of air bubbles in each microwell.
ER
EN
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8. Avoid bumping plates containing liquid reagents (especially
Working Conjugate Solution) to prevent adherence of liquid
to the plate sealer and/or top edges of the microwells.
R
EF
9. Adequate washing of the microwells with a validated
microplate washer is essential to eliminate non-specific
binding.
FO
R
10. Dry residue from the plate blocking process may be visible in
the microwells. Assay results will not be affected by this
material. Before reading the plates, carefully wipe the
bottom of the plates to remove any material that remains on
the outside of the wells, and ensure that all strips have been
pressed firmly into place.
11. For additional procedural instructions when running this
assay with the EVOLIS® Automated Microplate System,
consult the following documents:
•
EVOLIS® MATRM, Assay Module GS HIV-1/HIV-2
PLUS O EIA
•
EVOLIS® Operator’s Manual
20
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
12. For additional procedural instructions when running this
assay with the Elite™ Automated Microplate System, consult
the Elite™ User Manual or the Elite™ Microplate Assay
Testing Reference Manual for this assay.
13. For additional procedural instructions when running this
assay with the ORTHO® Summit System, refer to the
following documents:
ORTHO® Summit System User’s Guide
•
ORTHO® Summit Processor User’s Guide
•
ORTHO VERSEIA® User’s Guide
•
ORTHO VERSEIA® Integrated Processor User’s Guide
N
LY
•
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O
14. Prior to opening the plate pouch for the first time, verify that
some dark blue/purple granules remain inside the dessicant
pouch. A completely pink dessicant indicates this plate
pouch should not be used.
ER
EN
EIA Procedure
1. Perform equipment maintenance and calibration, where
necessary, as required by the manufacturer.
FO
R
R
EF
2. Bring all of the reagents except Conjugate Concentrate
to room temperature before beginning the assay
procedure.
3. Prepare Working Conjugate Solution, Working TMB
Solution, and Working Wash Solution if not previously
prepared. Mix gently by inversion. Mix again just before use.
4. Remove strips not needed for the assay and replace them
with labeled null strips, if necessary. Take care when
assembling partial plates with coated and uncoated (null)
strips, as automated systems cannot distinguish between
the strips and will report results for all wells that are assigned
a sample ID number (even if a null strip is inadvertently
placed where sample IDs have been assigned).
21
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
5. Microwell strips not needed for the assay may be returned to
the plate pouch and sealed, and then used at a later time.
Strips from different plates can only be mixed to assemble
full or partial plates if they are from the same plate lot and
have come from plates that have previously been tested with
kit controls and yielded valid runs. When assembling a plate
that contains strips from a newly opened, previously
untested plate, one of these strips should be placed at the
beginning of the plate and tested with the kit controls.
LY
6. If sample identity is not maintained by an automatic
procedure, identify the individual wells for each specimen or
control on a data sheet.
FO
R
R
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ER
EN
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SE
O
N
7. Dilute specimens and controls 3:4 in the Specimen
Diluent. Specimens and controls may be prediluted 3:4 in
the Specimen Diluent prior to addition of the diluted
specimen or control to the well (for example, dilute 150 μL of
specimen in 50 μL of Specimen Diluent and then transfer
100 μL to the well), or diluted in-well (add 25 μL of Specimen
Diluent to each well first, followed by 75 μL of specimen or
controls). Three Negative Controls, one HIV-1 Positive
Control, one HIV-2 Positive Control, and one HIV-1 Group O
Positive Control must be assayed on each plate or partial
plate of specimens. Mix each diluted specimen and control
thoroughly. Mix gently to avoid foaming of the diluent. All
microwell plates containing controls and specimens must be
subjected to the same process and incubation times.
NOTE: After adding the sample, the diluent will change from
purple to a blue color.
8. Add 100 μL of diluted specimen or control to the
appropriate wells of the microwell plate.
9. Cover the microwell plate with a plate sealer or use other
means to minimize evaporation. Incubate the plate for
60 ± 5 minutes at 37 ± 2°C.
10. At the end of the incubation period, carefully remove the
plate cover and aspirate the fluid from each well into a
22
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
biohazard container. Wash the microwell plate or strip a
minimum of five times with the Wash Solution (at least
400 μL/well/wash). Soak each well for 30 to 60 seconds
between each wash cycle. Aspirate the Wash Solution after
each wash. After the last wash, if excess liquid remains, blot
the inverted plate on clean, absorbent paper towels.
NOTE: Grasp the plate holder firmly at the center of the long
sides before inverting to blot.
O
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LY
11. Add 100 μL of Working Conjugate Solution (green
solution) to each well containing a specimen or control.
NOTE: Avoid bumping plates containing Working Conjugate
Solution to prevent contamination of the plate sealer and/or
top edges of the wells.
E
U
SE
12. Cover the microwell plate with a plate sealer or use other
means to minimize evaporation. Incubate the plate for
30 ± 5 minutes at 37 ± 2°C.
FO
R
R
EF
ER
EN
C
13. At the end of the incubation period, carefully remove the
plate cover and aspirate the fluid in each well into a
biohazard container. Wash the plate a minimum of five
times with Wash Solution (at least 400 μL/well/wash). Soak
each well for 30 to 60 seconds between each wash cycle.
Aspirate the Wash Solution after each wash. After the last
wash, if excess liquid remains, blot the inverted plate on a
clean, absorbent paper towel. NOTE: Grasp the plate holder
firmly at the center of the long sides before inverting to blot.
14. Add 100 μL of the Working TMB Solution to each well
containing a specimen or control. Incubate the plates in
the dark for 30 ± 5 minutes at room temperature (18 to
30°C). Use of a plate sealer or cover is optional.
15. Carefully remove the plate cover, if used, and add 100 μL of
Stopping Solution to each well to terminate the reaction.
Tap the plate gently, or use other means to assure
complete mixing. Complete mixing is required for
acceptable results.
23
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
16. Read absorbance within 30 minutes after adding the
Stopping Solution, using the 450 nm filter with 615 to
630 nm as reference. Ensure that all strips have been
pressed firmly into place before reading.
Decontamination
Dispose of all specimens and materials used to perform the test
as though they contain an infectious agent. Disposal should
comply with all applicable waste disposal requirements.
10-QUALITY CONTROL - VALIDATION OF RESULTS
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Mean Negative Control absorbance value (NCx)
Determine the mean absorbance for the Negative Control by
dividing the sum of the absorbance values by the number of
acceptable controls. The individual absorbance values of the
Negative Control must be greater than 0.000 AU and less than or
equal to 0.150 AU. One Negative Control absorbance value may
be discarded if it is outside this range. The NCx may be
calculated from the two remaining absorbance values.
0.080 (NCx)
FO
R
R
EF
Negative Control
Sample Number Absorbance Total Absorbance = 0.239 =
1
0.075
3
3
2
0.083
3
0.081
0.239
Calculation of Results:
Cutoff Value
Determine the cutoff value by adding the NCx to 0.250 as shown
in the example below:
NCx = 0.080
Cutoff Value = 0.080 + 0.250 = 0.330
24
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Assay Validation
A run is valid if the following criteria are met:
The absorbance values of the individual Negative Controls
are greater than 0.000 AU and less than or equal to 0.150
AU. One Negative Control value may be discarded. If two or
more Negative Controls are out of limit, the assay must be
repeated.
•
The absorbance value of the HIV-1 Positive Control must be
greater than or equal to 0.700 AU.
•
The absorbance value of the HIV-2 Positive Control must be
greater than or equal to 0.700 AU.
•
The absorbance value of the HIV-1 Group O Positive Control
must be greater than or equal to 0.700 AU.
SE
O
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•
C
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If any of these criteria have not been met, the assay is invalid and
must be repeated.
EN
11-INTERPRETATION OF RESULTS
EF
ER
The presence or absence of HIV Ab is determined by relating the
absorbance value of the specimen to the cutoff value.
FO
R
R
Specimens with absorbance values that are < 0.000 must be
repeated. Those with values greater than the upper linearity
limits of the reader should be reported as reactive.
Specimens with absorbance values less than the cutoff value are
considered non-reactive by the GS HIV-1/HIV-2 PLUS O EIA and
may be considered negative for HIV-1 (M and O Groups) and
HIV-2 antibodies. Further testing is not required.
Specimens with absorbance values greater than or equal to the
cutoff value are considered initially reactive by the GS
HIV-1/HIV-2 PLUS O EIA. Initially reactive specimens should be
retested in duplicate to validate the initial test results. Repeat
testing of intially reactive specimens processed manually or with
the ORTHO® Summit System should be retested either manually
or with the ORTHO® Summit System. If, after repeat testing, the
25
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
absorbance values of both duplicate specimens are less than the
cutoff value, the original specimen may be considered
non-repeatedly reactive and negative for HIV-1 (Groups M and
O) and HIV-2 antibodies.
If, after repeat testing, the absorbance value of either of the
duplicates is greater than or equal to the cutoff value, the
specimen must be considered repeatedly reactive.
U
SE
O
N
LY
If the specimen is repeatedly reactive, the probability that
antibodies to HIV-1 and/or HIV-2 are present is high, especially
for specimens obtained from subjects at increased risk for HIV-1
and/or HIV-2 infection, or for specimens with very high
absorbance values. In most settings, it is appropriate to
investigate repeatedly reactive specimens by additional, more
specific or supplemental tests, such as Western blot or
immunofluorescence.
Specimens that are repeatedly reactive by the GS
HIV-1/HIV-2 PLUS O EIA and are found to be positive for
antibodies to HIV-1 by additional, more specific or
supplemental testing but negative or indeterminate for
antibodies to HIV-2 are considered to be positive for
antibodies to HIV-1.
•
Specimens that are repeatedly reactive by the GS
HIV-1/HIV-2 PLUS O EIA and are found to be positive by
additional, more specific or supplemental testing for
antibodies to HIV-2, but negative or indeterminate for
antibodies to HIV-1, are considered to be positive for
antibodies to HIV-2.
•
Specimens that are repeatedly reactive by the GS
HIV-1/HIV-2 PLUS O EIA and are found to be positive by
additional, more specific or supplemental testing for both
HIV-1 and HIV-2 antibodies may contain antibodies that
cross-react with both virus types, or may be indicative of a
dual infection with both HIV-1 and HIV-2.
FO
R
R
EF
ER
EN
C
E
•
26
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
•
The interpretation of results of specimens found to be
repeatedly reactive by GS HIV-1/HIV-2 PLUS O EIA and
negative or indeterminate on additional, more specific
testing for antibodies to both HIV-1 and HIV-2 is unclear.
Clarification may sometimes be obtained by testing another
specimen taken three to six months later.
12-LIMITATIONS OF THE PROCEDURE
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1. The GS HIV-1/HIV-2 PLUS O EIA Procedure and the
Interpretation of Results must be followed closely when
testing for the presence of antibodies to HIV-1 and/or HIV-2
in plasma, serum, or cadaveric serum specimens. The user
of this kit is advised to read the package insert carefully prior
to conducting the test. In particular, the test procedure must
be carefully followed for sample and reagent pipetting, plate
washing, and time and temperature of the incubation steps.
Testing of other body specimens, pooled blood or
processed plasma, and products made from such pools is
not recommended.
FO
R
R
EF
2. The GS HIV-1/HIV-2 PLUS O EIA detects circulating
antibodies to HIV-1 (Groups M and O) and HIV-2 and thus is
useful in screening blood and plasma donated for
transfusion and further manufacture, in screening cadaveric
serum for tissue donation, in evaluating patients with signs
or symptoms of AIDS, and in establishing prior infection with
HIV-1 or HIV-2. Clinical studies continue to clarify and refine
the interpretation and medical significance of the presence
of antibodies to HIV-1 or HIV-2.42 Repeatedly reactive
specimens must be investigated by additional, more
specific, or supplemental tests. Recommendations for
appropriate use of such additional tests may be issued
periodically by the United States Public Health Service. For
individuals who are confirmed positive for antibodies,
appropriate counseling and medical evaluation should be
offered. Both confirmation of the test result on a freshly
27
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
drawn sample and counseling should be considered an
important part of testing for antibody to HIV-1 and HIV-2.
3. AIDS and AIDS-related conditions are clinical syndromes
and their diagnosis can only be established clinically.42
Testing alone cannot be used to diagnose AIDS, even if the
recommended investigation of reactive specimens suggests
a high probability that the antibody to HIV-1 or HIV-2 is
present.
LY
4. A negative test result at any point in the investigation of
individual subjects does not preclude the possibility of
exposure to or infection with HIV-1 and/or HIV-2.
U
SE
O
N
5. Negative results can occur if the quantity of marker present
in the sample is too low for the detection limits of the assay,
or if the marker which is detected is not present during the
stage of disease in which a sample is collected.
ER
EN
C
E
6. Failure to add specimen or reagent as instructed in the
procedure could result in a falsely negative test. Repeat
testing should be considered where there is clinical
suspicion of infection or procedural error.
FO
R
R
EF
7. The risk of an asymptomatic person with a repeatedly
reactive test developing AIDS or an AIDS-related condition is
not known, as the course of HIV infection may vary among
individual patients and may be altered by antiretroviral
therapy.43,44 However, in a prospective study, AIDS
developed in 51% of homosexual men after 10 years of
infection.45
8. Data obtained from testing persons both at increased and at
low risk for HIV-1 and/or HIV-2 infection suggest that
repeatedly reactive specimens with high reactivity on the GS
HIV-1/HIV-2 PLUS O EIA may be more likely to demonstrate
the presence of antibodies to HIV-1 (Groups M and O)
and/or HIV-2 by additional, more specific, or supplemental
testing.46 Borderline reactivity is more frequently nonspecific,
especially in samples obtained from persons at low risk for
infection with HIV-1 or HIV-2; however, the presence of
28
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
antibodies to HIV-1 and/or HIV-2 in some of these
specimens can be demonstrated by additional, more
specific, or supplemental testing, or by testing a subsequent
sample drawn at a later date (e.g. 3 to 6 months).47
9. It is generally recognized that detection of HIV antibody in
infants born to seropositive mothers is not adequate to
diagnose HIV infection in the infant, since maternal IgG
frequently persists for as long as 18 months after birth.
Supplemental assays designed specifically for neonatal
specimens may be helpful in resolving such cases.48
O
N
LY
10. An absorbance value of less than 0.000 AU may indicate a
procedural or instrument error that should be evaluated.
That result is invalid and that specimen must be re-run.
ER
EN
C
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11. Factors that can affect the validity of results include failure to
add the specimen or reagents to the well, inadequate
washing of microplate wells, failure to follow stated
incubation times and temperatures, addition of wrong
reagents to wells, the presence of metals, or splashing of
bleach into wells.
EF
12. Non-repeatedly reactive specimens can be caused by:
improper washing of microwell plates during the initial
test
•
cross-contamination of nonreactive specimens with HIV
antibody from a high-titered specimen
•
contamination of the Chromogen or Working TMB
Solution by oxidizing agents (sodium hypochlorite,
hydrogen peroxide, etc.)
•
contamination of the Stopping Solution
FO
R
R
•
13. A person who has antibodies to HIV-1 is presumed to be
infected with the virus, except that a person who has
participated in an HIV vaccine study may develop antibodies
to the vaccine and may or may not be infected with HIV.
Clinical correlation is indicated with appropriate counseling,
29
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
medical evaluation, and possibly additional testing to decide
whether a diagnosis of HIV infection is accurate.
13-PERFORMANCE CHARACTERISTICS OF SERUM
AND PLASMA TESTING
N
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Reproducibility—Manual Testing
A panel of 14 specimens was used for determining the
reproducibility of the GS HIV-1/HIV-2 PLUS O EIA. The 14
member panel included 5 dilutions of an HIV-1 antibody positive
sample (Group M); one HIV-1 (Group O) antibody positive
sample (rabbit); 6 dilutions of an HIV-2 antibody positive sample;
and two undiluted HIV-negative samples.
#
8
9
10
11
12
13
14
E
C
EN
ER
HIV-1 Positive
HIV-1 Positive
HIV-1 Group O Positive
HIV-1 Low Positive
HIV-1 High Negative
HIV Negative
HIV-2 Positive
Panel Member Composition
HIV-2 Positive
HIV-2 Positive
HIV-2 Low Positive
HIV-2 High Negative
HIV-2 Negative
HIV Negative
HIV Negative
U
Panel Member Composition
EF
#
1
2
3
4
5
6
7
SE
O
The composition of the panel was as follows:
FO
R
R
The specimens were tested in triplicate on 3 different days using
3 different test kit lots at each of 6 sites. The data were analyzed
according to the principles described in the Clinical and
Laboratory Standards Institute guidance (CLSI) EP15-A2. The
standard deviation (SD) and percent coefficient of variation
(%CV) were calculated from the observed Optical Density (OD)
and Signal to Cutoff values, respectively, for each panel member
and are presented in the tables below.
30
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 1a: Reproducibility of the GS HIV-1/HIV-2 PLUS O EIA by Optical
Density (OD) values
Between Day
Between Site
Total
Na
Mean
(OD)
SD
%CV
SD
%CV
SD
%CV
SD
1
162
1.240
0.113
9.1
0.039
3.2
0.000
0.0
0.120
9.7
2
162
0.972
0.081
8.3
0.047
4.8
0.052
5.3
0.107
11.0
3
162
0.485
0.116
23.9
0.000
0.0
0.080
16.5
0.141
29.0
4
161
0.423
0.042
10.0
0.019
4.4
0.013
3.1
0.048
11.4
5
162
0.255
0.028
11.1
0.016
6.1
0.021
8.2
0.039
15.1
6
161
0.128
0.021
16.8
0.008
6.3
0.008
6.0
0.024
18.9
7
162
1.195
0.132
11.1
0.066
5.5
0.130
10.9
0.197
16.5
8
162
0.845
0.103
12.2
0.037
4.4
0.113
13.3
0.157
18.6
9
162
0.648
0.089
13.7
0.031
4.8
0.068
10.5
0.116
17.9
10
162
0.401
0.042
10.4
0.026
6.5
0.052
13.1
0.072
18.0
11
162
0.236
0.024
10.1
0.016
6.7
0.028
12
162
0.112
0.012
10.3
0.007
6.4
0.011
LY
Within Run
Panel
Member
13
162
0.041
0.006
14.4
0.004
8.9
0.008
14
162
0.044
0.009
21.5
0.002
5.4
0.008
0.040
9.7
N
0.017
15.5
19.0
0.010
25.4
17.4
0.012
28.2
SE
O
12.0
17.0
U
a. Outliers not included in statistical calculations.
%CV
EN
C
E
Table 1b: Reproducibility of the GS HIV-1/HIV-2 PLUS O EIA by Signal to
Cutoff (S/CO) values
Within Run
Na
Mean
(S/CO)
SD
1
162
4.09
0.370
2
162
3.21
0.254
3
162
1.60
4
161
1.40
5
162
0.84
6
161
7
Between Day
Between Site
Total
SD
%CV
SD
%CV
SD
9.0
0.125
3.1
0.035
0.9
0.392
9.6
7.9
0.139
4.3
0.220
6.9
0.364
11.3
0.373
23.3
0.000
0.0
0.280
17.5
0.466
29.1
10.1
0.048
3.5
0.048
3.4
0.156
11.2
0.085
10.2
0.044
5.2
0.072
8.6
0.120
14.3
0.42
0.069
16.3
0.029
6.9
0.014
3.4
0.076
18.1
162
3.94
0.452
11.5
0.163
4.1
0.422
10.7
0.640
16.3
8
162
2.79
0.362
13.0
0.077
2.8
0.362
13.0
0.518
18.6
9
162
2.14
0.292
13.7
0.087
4.1
0.232
10.8
0.383
17.9
10
162
1.32
0.145
11.0
0.070
5.3
0.169
12.8
0.233
17.7
11
162
0.78
0.083
10.7
0.043
5.6
0.092
11.8
0.131
16.9
12
162
0.37
0.037
10.1
0.018
5.0
0.032
8.7
0.053
14.3
13
162
0.14
0.018
13.0
0.011
8.0
0.024
17.7
0.032
23.4
14
162
0.14
0.029
20.1
0.007
4.7
0.023
15.9
0.037
26.0
EF
ER
%CV
R
Panel
Member
FO
R
0.141
a. Outliers not included in statistical calculations.
31
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
%CV
Reproducibility and Precision Testing—EVOLIS® Automated
Microplate System
A panel of 15 specimens was used for determining the
reproducibility and precision of the GS HIV-1/HIV-2 PLUS O EIA.
The 15 member panel included 6 serum members (5 positive and
1 negative), 5 plasma members (4 positive and 1 negative), and
the 4 GS HIV-1/HIV-2 PLUS O EIA kit controls. The composition
of the panel was as follows:
N
LY
Panel Member Composition
HIV-1 Group O Positive (Serum)
HIV High Negative (Serum)
HIV High Negative (Plasma)
HIV-1 Positive Control
HIV-2 Positive Control
HIV-1 Group O Positive Control
HIV Negative Control
O
#
9
10
11
12
13
14
15
SE
Panel Member Composition
HIV-1 Moderate Positive (Serum)
HIV-1 Moderate Positive (Plasma)
HIV-1 Low Positive (Serum)
HIV-1 Low Positive (Plasma)
HIV-2 Moderate Positive (Serum)
HIV-2 Moderate Positive (Plasma)
HIV-2 Low Positive (Serum)
HIV-2 Low Positive (Plasma)
U
#
1
2
3
4
5
6
7
8
FO
R
R
EF
ER
EN
C
E
Reproducibility
Reproducibility of the GS HIV-1/HIV-2 PLUS O EIA was
determined for the EVOLIS® Automated Microplate System by
testing the panel at three (3) sites on three (3) different EVOLIS®
instruments (one at each site) using a single lot of the GS
HIV-1/HIV-2 PLUS O EIA. Each panel member was tested in
triplicate (x3) on 2 runs per day for 5 days (30 replicates per
member per lot/site). The data were analyzed according to the
principles described in the Clinical and Laboratory Standards
Institute guidance (CLSI) EP15-A2. The standard deviation (SD)
and percent coefficient of variation (%CV) were calculated for
each panel member and are presented in the table below.
32
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 2: Reproducibility Results - EVOLIS® Testing By Signal to Cutoff Ratio
(S/CO) N = 90 for each panel membera
Between Run
Between Day
Between Site
Totalb
SD
%CV
SD
%CV
SD
%CV
SD
%CV
SD
%CV
1
2.74
0.114
4.2
0.160
5.8
0.110
4.0
0.116
4.2
0.254
9.3
2
2.34
0.064
2.8
0.120
5.1
0.059
2.5
0.031
1.3
0.151
6.5
3
1.49
0.063
4.2
0.049
3.3
0.045
3.0
0.049
3.3
0.104
7.0
4
1.36
0.055
4.1
0.065
4.8
0.044
3.3
0.068
5.0
0.118
8.7
5
2.19
0.102
4.7
0.299
13.6
0.232
10.6
0.221
10.1
0.450
20.5
6
2.34
0.247
10.6
0.358
15.3
0.057
2.4
0.384
16.4
0.583
24.9
7
1.64
0.056
3.4
0.242
14.8
0.135
8.2
0.177
10.8
0.334
20.4
8
1.67
0.097
5.8
0.309
18.5
0.105
6.3
0.277
16.6
0.439
26.3
9
2.12
0.105
5.0
0.246
11.6
0.166
7.8
0.149
7.0
0.348
16.4
10
0.71
0.028
3.9
0.040
5.6
0.000
0.0
0.012
1.7
0.050
7.0
11
0.75
0.036
4.9
0.032
4.4
0.015
2.0
3.9
0.058
7.8
12
7.10
0.187
2.6
0.434
6.1
0.164
2.3
0.353
5.0
0.612
8.6
13
6.13
0.186
3.0
0.746
12.2
0.617
10.1
0.462
7.5
1.089
17.8
14
6.49
0.233
3.6
0.419
6.5
0.284
4.4
0.484
7.5
0.738
11.4
15
0.21
0.023
11.1
0.041
19.9
0.000
0.0
0.024
11.5
0.053
25.5
N
LY
Mean
(S/CO)
U
Within Run
Panel
Member
E
SE
O
0.029
ER
EN
C
a One (1) result was missing for panel member 2 due to insufficient sample volume.
b Total: Total variability of the assay performance includes within run, between run, between day, and between
site.
FO
R
R
EF
Precision
Precision of the GS HIV-1/HIV-2 PLUS O EIA was determined for
the EVOLIS® Automated Microplate System by testing the panel
at one (1) site on one (1) EVOLIS® instrument using a single lot of
the GS HIV-1/HIV-2 PLUS O EIA. Each panel member was
tested in duplicate (x2) on two runs per day for a period of 20
days (80 replicates per member). The data were analyzed at
Bio-Rad Laboratories according to the principles described in
the Clinical and Laboratory Standards Institute guidance (CLSI)
EP5-A2. The standard deviation (SD) and percent coefficient of
variation (%CV) were calculated for each panel member and are
presented in the table below.
33
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 3: Precision Results - EVOLIS® Testing By Signal to Cutoff Ratio
(S/CO) N = 80
Within Run
Between Run
Totala
Between Day
Mean
(S/CO)
SD
%CV
SD
%CV
SD
%CV
SD
%CV
1
2.84
0.071
2.5
0.090
3.2
0.065
2.3
0.131
4.6
2
2.34
0.062
2.6
0.075
3.2
0.052
2.2
0.110
4.7
3
1.49
0.043
2.9
0.062
4.2
0.036
2.5
0.084
5.7
4
1.49
0.063
4.2
0.048
3.2
0.058
3.9
0.098
6.6
5
1.97
0.065
3.3
0.214
10.9
0.181
9.2
0.288
14.6
6
1.85
0.218
11.8
0.258
14.0
0.161
8.7
0.374
20.3
7
1.51
0.054
3.6
0.177
11.7
0.000
0.0
0.185
12.3
8
1.38
0.052
3.8
0.184
13.3
0.000
0.0
0.191
13.8
9
1.93
0.061
3.2
0.232
12.0
0.000
0.0
0.240
12.4
10
0.75
0.026
3.5
0.031
4.1
0.027
3.6
0.049
6.5
11
0.75
0.026
3.5
0.032
4.3
0.010
1.4
0.043
5.7
12
7.73
0.202
2.6
0.234
3.0
0.331
4.3
13
5.87
0.192
3.3
0.428
7.3
0.000
14
6.32
0.155
2.4
0.444
7.0
0.000
15
0.24
0.029
12.2
0.046
19.4
0.000
N
LY
Panel
Member
5.9
0.469
0.0
0.470
7.4
0.0
0.054
22.9
U
0.0
SE
O
0.453
8.0
C
E
a Total: Total variability of the assay performance includes within run, between run, and between day.
FO
R
R
EF
ER
EN
Reproducibility and Precision Testing - Elite™ Automated
Microplate System
The reproducibility and precision of the Elite™ Automated
Microplate System with the GS HIV-1/HIV-2 PLUS O EIA were
determined using a panel of 17 members that consisted of 7
serum, 6 plasma, and 4 kit controls. The composition of the
panel is as follows:
#
1
2
3
4
5
6
7
8
9
Panel Member Composition
HIV-1 Moderate Positive Plasma
HIV-1 Low Positive Plasma
HIV-2 Moderate Positive Plasma
HIV-2 Low Positive Plasma
Low Negative Plasma
HIV-1 High Negative Plasma
HIV-1 Moderate Positive Serum
HIV-1 Low Positive Serum
HIV-2 Moderate Positive Serum
#
10
11
12
13
14
15
16
17
34
Panel Member Composition
HIV-2 Low Positive Serum
HIV-1 Group O Moderate Positive Serum
Low Negative Serum
HIV-1 High Negative Serum
HIV-1 High Positive Control
HIV-2 High Positive Control
HIV-1 Group O High Positive Control
Low Negative Control
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Reproducibility
Reproducibility testing of the GS HIV-1/HIV-2 PLUS O EIA was
performed on the Elite™ Automated Microplate System at 3
sites using 3 different Elite™ instruments (one at each site), and
1 lot of the GS HIV 1/HIV-2 PLUS O EIA. Each panel member
was tested in triplicate on 2 runs per day for 5 days (30 replicates
per member per site). The data were analyzed according to the
principles described in the CLSI guidance EP15-A2. The SD and
%CV that were calculated for each panel member are shown in
the table below.
Between Run
Between Day
Mean
S/CO
SD
%CV
SD
%CV
SD
%CV
1
90
2.56
0.130
5.1
0.086
3.4
0.023
0.9
2
90
1.63
0.051
3.1
0.051
3.1
0.044
3
90
2.11
0.233
11.0
0.141
6.7
0.026
4
90
1.61
0.199
12.4
0.114
7.1
5
90
0.13
0.019
14.8
0.000
0.0
6
90
0.96
0.052
5.4
0.029
3.1
7
88
2.52
0.058
2.3
0.107
8
90
1.55
0.051
3.3
9
88
2.37
0.158
6.7
10
87
1.64
0.117
7.2
11
89
1.97
0.144
12
90
0.11
0.012
13
89
0.75
0.026
14
90
6.44
15
90
16
17
SD
%CV
0.150
5.9
0.218
8.5
2.7
0.093
5.7
0.126
7.7
1.2
0.146
6.9
0.309
14.7
0.000
0.0
0.166
10.3
0.283
17.6
0.015
11.9
0.021
16.5
0.032
25.2
0.016
1.6
0.051
5.4
0.080
8.4
4.2
0.029
1.2
0.139
5.5
0.187
7.4
0.050
3.2
0.042
2.7
0.113
7.3
0.140
9.0
0.241
10.2
0.000
0.0
0.093
3.9
0.302
12.8
0.203
12.4
0.077
4.7
0.134
8.2
0.281
17.2
0.125
6.4
0.074
3.8
0.000
0.0
0.205
10.4
O
SE
E
EN
ER
0.000
0.0
0.014
13.5
0.033
31.2
0.038
35.9
3.4
0.030
4.0
0.022
2.9
0.022
2.9
0.050
6.7
0.225
3.5
0.238
3.7
0.000
0.0
0.337
5.2
0.471
7.3
5.60
0.308
5.5
0.467
8.3
0.000
0.0
0.190
3.4
0.591
10.6
90
4.96
0.209
4.2
0.259
5.2
0.181
3.6
0.000
0.0
0.379
7.7
89
0.12
0.023
18.9
0.000
0.0
0.014
11.3
0.008
6.3
0.028
22.9
FO
R
11.5
SD
Totala
%CV
EF
R
7.3
Between Site
U
#
C
Panel
Member
N
Within Run
LY
Table 4: Reproducibility Results - Elite™ Testing by Signal to Cutoff Ratio (SCO)
a. Total: Total variability of the assay performance includes within run, between run, between day, and between site.
Precision
Precision testing of the GS HIV-1/HIV-2 PLUS O EIA with the
Elite™ Automated Microplate System was performed with one
instrument at one site. The 17-member panel was tested in
duplicate on two runs per day for 12 days on 1 lot of the GS
HIV-1/HIV-2 PLUS O EIA assay (total number of replicates per
each assay = 2 (in duplicate) x 2 runs/day x 12 days = 48
35
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
replicates per panel member). The data were analyzed
according to the principles described in the CLSI guidance
EP5-A2. The SD and %CV that were calculated for each panel
member are shown in the table below.
Table 5: Precision Results - Elite™ Testing by Signal to Cutoff Ratio (SCO)
%CV
SD
%CV
SD
%CV
SD
%CV
1
48
2.87
0.124
4.3
0.053
1.9
0.094
3.3
0.164
5.7
2
48
1.77
0.041
2.3
0.055
3.1
0.020
1.1
0.072
4.1
3
48
2.03
0.128
6.3
0.126
6.2
0.095
4.7
0.203
10.0
4
48
1.42
0.104
7.3
0.140
9.9
0.000
0.0
0.175
12.3
5
48
0.12
0.013
10.7
0.005
4.4
0.022
18.6
0.026
21.9
6
48
1.00
0.040
4.0
0.025
2.5
0.023
2.3
0.052
5.2
7
47*
2.66
0.059
2.2
0.038
1.4
0.064
2.4
0.094
3.6
8
47*
1.68
0.034
2.0
0.034
2.0
0.048
2.9
0.068
4.0
9
48
2.37
0.150
6.4
0.205
8.7
0.000
0.0
0.254
10.8
10
48
1.65
0.136
8.2
0.166
10.0
0.000
0.214
13.0
47*
1.87
0.083
4.4
0.177
SE
0.0
11
9.5
0.136
7.3
0.238
12.8
12
48
0.12
0.019
16.3
0.000
0.0
0.032
27.3
0.037
31.8
13
47*
0.85
0.022
2.6
0.026
3.1
0.067
7.9
0.075
8.9
14
48
6.90
0.166
2.4
0.189
2.7
0.062
0.9
0.259
3.8
15
48
5.91
0.258
4.4
0.595
10.1
0.079
1.3
0.653
11.0
16
48
5.12
0.115
2.2
0.240
4.7
0.309
6.0
0.408
8.0
17
48
0.16
0.020
12.8
0.000
0.0
0.036
23.4
0.041
26.6
C
EN
ER
EF
O
N
LY
Between Day
SD
U
Mean
S/CO
Between Run
N
E
Within Run
Totala
Panel
Member
FO
R
R
a. Total: Total variability of the assay performance includes within run, between run, between site, and between days.
36
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
SE
O
N
LY
SENSITIVITY AND SPECIFICITY
Testing to determine the sensitivity and specificity of the GS
HIV-1/HIV-2 PLUS O EIA was performed manually, and
specificity was also determined with the ORTHO® Summit
System (using both the ORTHO® Summit Sample Handling
System* and the ORTHO VERSEIA® Pipetter). In addition,
comparative sensitivity and specificity studies were used to
evaluate use of the EVOLIS® and Elite™ Automated Microplate
Systems, and comparative sensitivity studies were used to
evaluate the ORTHO® Summit System. Unless otherwise noted
in the tables that follow, the results summarize the manual
testing that was performed. The results of the testing with the
EVOLIS®, Elite™, and ORTHO® Summit Systems are described
in text, where applicable.
FO
R
R
EF
ER
EN
C
E
U
Specificity Studies
Reactivity in Random Blood Donors and Individuals with
Medical Conditions Unrelated to HIV-1 or HIV-2
The results of testing specimens from random blood and plasma
donors and specimens from individuals with medical conditions
unrelated to HIV-1 or HIV-2 infection with the GS HIV-1/HIV-2
PLUS O EIA are summarized in Tables 6 and 7. The data include
serum and plasma samples obtained from donors at 3
geographically distinct locations, and 360 specimens from
individuals with various medical conditions.
*
The ORTHO® Summit Sample Handling System is now a legacy device and no longer available for marketing.
37
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 6: Detection of Antibodies to HIV-1 and/or HIV-2 in Random Donors
Results Obtained with
GS HIV-1/HIV-2 PLUS O EIA
Group
(Sample Type = Serum)
Repeatedly Reactive
Specimens
HIV-2 EIA
Repeatedly
Reactive
Pos. by HIV-1
Immunoblot
alone
3
(0.10%)
0
0
3
(0.10%)
3
(0.10%)
0
0
6
(0.10%)
6
(0.10%)
0
0
Number
Tested
NonReactive
Initially
Reactive
Repeatedly
Reactive
Random Blood
Donors, Site 1
2999
(100.00%)
2996
(99.90%)
3
(0.10%)
Random Blood
Donors, Site 2
3104
(100.00%)
3101
(99.90%)
SUB TOTAL:
6103
(100.00%)
6097
(99.90%)
Results Obtained with
GS HIV-1/HIV-2 PLUS O EIA
Pos. by HIV-1
Immunoblot
alone
0
0
2
(0.09%)
0
0
6
(0.12%)
0
0
12
(0.11%)
0
0
Initially
Reactive
Repeatedly
Reactive
Random Blood
Donors, Site 2
2901
(100.00%)
2895
(99.79%)
6
(0.21%)
4
(0.14%)
Random Blood
Donors, Site 3
2155
(100.00%)
2149
(99.72%)
6
(0.28%)
SUB TOTAL:
5056
(100.00%)
5044
(99.76%)
12
(0.24%)
TOTAL:
(Serum and Plasma)
11,159
(100.00%)
11,141
(99.84%)
C
E
U
O
N
NonReactive
ER
EN
18
(0.16%)
LY
HIV-2 EIA
Repeatedly
Reactive
Number
Tested
SE
Group
(Sample Type = Plasma)
Repeatedly Reactive
Specimens
FO
R
R
EF
As shown in Table 6, 99.84% of the normal donor population
(n = 11,159) were initially nonreactive, 0.16% were initially
reactive, and 0.11% were repeatedly reactive. Twelve of the 18
initially reactive specimens were repeatedly reactive upon
retesting. None of the repeatedly reactive specimens were
positive for antibodies to HIV-1 or HIV-2 by Western blot.
Specificity of the GS HIV-1/HIV-2 PLUS O EIA was estimated
from the results of screening tests in random blood and plasma
donors, and determined by the following formula:
(# normal donor specimens - # repeatedly reactive specimens)
X 100
(# normal donor specimens - repeatedly reactive specimens
confirmed positive for antibodies to HIV)
Thus, assuming a zero prevalence rate of antibodies to HIV-1
and HIV-2 in this population, the GS HIV-1/HIV-2 PLUS O EIA
38
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
had an estimated specificity in this study of (11,159 – 12) x
100/11,159 = 99.89% (95% confidence interval: 99.83 – 99.96).
EN
C
E
U
SE
O
N
LY
Reactivity of Known Negatives Using the EVOLIS®
Automated Microplate System
Comparative studies using the EVOLIS® Automated Microplate
System in comparison to manual testing with the GS
HIV-1/HIV-2 PLUS O EIA were performed with specimens known
to be negative for HIV antibody. A total of 100 known negative
samples were tested once on each of three different instruments
at each of three different sites. Approximately 30-40% of the
known negative samples were near the assay cut-off. For the
100 known negative samples combined across sites, 99.7%
(299/300) of manual results were nonreactive; 100% (300/300) of
the EVOLIS® Automated Microplate System results were
nonreactive. The negative % agreement was 100% (299/299)
with a 95% confidence interval range of 98.7%-100.0%. These
studies demonstrate comparable results for both methods when
testing with the GS HIV-1/HIV-2 PLUS O EIA.
FO
R
R
EF
ER
Reactivity of Known Negatives Using the Elite™ Automated
Microplate System
The performance of the Elite™ Automated Microplate System
was compared to manual testing of the GS HIV-1/HIV-2 PLUS O
EIA with specimens known to be negative for HIV antibody. A
panel of 100 known negative samples was tested once on each
of three different instruments at each of three different sites.
Approximately 30-40% of the known negative samples were
near the assay cut-off. All of the 100 known negative samples
were nonreactive at all 3 sites, with both the Elite™ Automated
Microplate System and manual testing. Averaged across all
sites, the overall % agreement was 100% (100/100) with a 95%
confidence interval range of 96.4%-100.0%. These studies
demonstrate comparable results for both methods when testing
with the GS HIV-1/HIV-2 PLUS O EIA.
39
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
U
SE
O
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LY
Random Blood Donors Tested with ORTHO® Summit System
Additional specificity studies have been performed with the
GS HIV-1/HIV-2 PLUS O EIA using the ORTHO® Summit System
with the ORTHO® Summit Sample Handling System.* In total,
24,250 normal donors (including a combination of serum and
plasma specimens) were tested at 3 U.S. blood centers. Three
(3) samples that were repeatedly reactive and confirmed positive
by HIV-1 Western blot were excluded from the specificity
analysis. Of the remaining 24,247 samples tested, 52 were
initially reactive (0.21%) and 2 of these specimens were QNS for
repeat testing or confirmation. There were 17 repeatedly reactive
specimens that were either negative (6), indeterminate (9), or not
tested (2) by Western blot. Therefore, the GS HIV-1/HIV-2
PLUS O EIA had an estimated specificity in this study of 99.92%
(24,228/24,247; 95% confidence interval: 99.88 – 99.96).
EF
ER
EN
C
E
Specificity studies have also been performed with unlinked
random donor serum and plasma specimens on the ORTHO®
Summit System using the ORTHO VERSEIA® Pipetter. The
combined testing at 3 testing sites showed a specificity of 100%
(3216/3216; 95% confidence interval: 99.88 – 100.0%).
FO
R
R
Additional studies have also been performed with unlinked
random donor serum and plasma specimens with the standalone ORTHO VERSEIA® Pipetter and a stand-alone ORTHO®
Summit Processor (OSP) in comparison to the ORTHO
VERSEIA® Integrated Processor (VIP). Both the VIP system and
the stand-alone system showed a specificity of 100%
(2019/2019; 95% confidence interval: 99.82 – 100.0%). The VIP
S/CO percent difference increased from the stand-alone
VERSEIA®/stand-alone OSP by 4.6%.
*
The ORTHO® Summit Sample Handling System is now a legacy device no longer
available for marketing.
40
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 7: Detection of Antibodies to HIV-1 and/or HIV-2 in Individuals with
Other Medical Conditions Unrelated to HIV Infection
Results Obtained with
GS HIV-1/HIV-2 PLUS O EIA
Repeatedly Reactive
Specimens
HIV-2 EIA
Repeatedly
Reactive
Pos. by HIV-1
Immunoblot
alone
2
(2.50%)
0
0
1
(0.50%)
1
(0.50%)
0
0
20
(100.00%)
0
(0.00%)
0
(0.00%)
NA
NA
60
(100.00%)
59
(98.33%)
1
(1.67%)
1
(1.67%)
0
0
360
(100.00%)
356
(98.89%)
4
(1.11%)
4
(1.11%)
NonReactive
Initially
Reactive
Repeatedly
Reactive
Immunological
diseasesa
80
(100.00%)
78
(97.50%)
2
(2.50%)
Acute or chronic
viral diseases/
parasitic diseasesb
200
(100.00%)
199
(99.50%)
Malignanciesc
20
(100.00%)
Miscellaneous/
Otherd
TOTAL:
0
0
O
N
Number
Tested
LY
Group
EN
C
E
U
SE
a. 20 SLE (ANA positive); 20 Rheumatoid arthritis (RF positive); 20 IgG Hypergammaglobulinemia; 20 IgM
Hypergammaglobulinemia
b. 20 HCV; 20 HBV; 20 HAV; 20 CMV; 20 EBV; 20 HSV; 20 HTLV-I/II; 20 Rubella; 20 Syphilis; 20 Toxoplasmosis
c. 2 Adenocarcinoma; 2 Bladder Cancer; 2 Breast Cancer; 3 Colon Cancer; 1 Endometrial Cancer; 1 Lung
Cancer; 1 Melanoma, metastatic; 3 Prostate Cancer; 3 Rectal Cancer; 1 Renal Cell Cancer; 1 Squamous Cell
Cancer
d. 20 Non Viral Cirrhosis [(primary biliary (5); alcohol induced (8); drug induced (7)]; 20 Multiple Transfusions; 20
Multiparous females
FO
R
R
EF
ER
Four specimens from individuals with unrelated medical
conditions were initially and repeatedly reactive in the GS
HIV-1/HIV-2 PLUS O EIA. Of the 4 specimens, 1 was from an
individual with cirrhosis; 1 was from an individual with HCV; and
2 were from individuals positive for Rheumatoid Factor (RF).
Three of the 4 specimens were negative and 1 was indeterminate
when tested with a licensed HIV-1 Western Blot. All of the
specimens were nonreactive for antibody to HIV-2 when tested
with a licensed HIV-2 EIA. None of the remaining specimens
from individuals with other medical conditions were reactive in
the GS HIV-1/HIV-2 PLUS O EIA. There appears to be no
correlation between reactivity in the GS HIV-1/HIV-2 PLUS O EIA
and other medical conditions unrelated to HIV infection.
41
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Sensitivity Studies
Reactivity in Specimens Known to be Positive for Antibodies
to HIV-1
The reactivity of the GS HIV-1/HIV-2 PLUS O EIA was
determined by testing serum and plasma samples from patients
diagnosed as having AIDS (n = 313), and from 689 individuals
known to be HIV-1 antibody positive from U.S. (n = 490) and
non-U.S. locations (n = 199a) for whom the clinical status was
unknown. The results of testing are shown in Table 8.
Table 8: Reactivity in HIV-1 Known Positive Specimens
313 (100%)
Known HIV-1 Positive U.S.
(N = 490)
490 (100%)
313 (100%)
1002 (100%)
490 (100%)
199 (100%)
1002 (100%)
EN
C
Nigeria (N = 46)
Sierra Leone (N = 40)
Thailand (N = 21)
Zimbabwe (N = 8)
ER
a. Australia, New S. Wales (N = 36)
Central African Republic (N = 40)
Ghana (N = 5)
Kenya (N = 3)
U
199 (100%)
TOTAL
E
Known HIV-1 Positive non U.S.
(N = 199a)
SE
O
AIDS
(N = 313)
Licensed HIV-1/HIV-2 EIA
No. Repeatedly Reactive
LY
GS HIV-1/HIV-2 PLUS O EIA
No. Repeatedly Reactive
N
Group
FO
R
R
EF
Of the 313 diagnosed AIDS patients, 100% were repeatedly
reactive with the GS HIV-1/HIV-2 PLUS O EIA. All AIDS
specimens were positive on a licensed HIV-1 Western blot. Of
the known 689 positives from U.S. and non-U.S. locations, all
were confirmed positive with one of four licensed HIV-1 Western
blots.
The HIV-1 sensitivity of the GS HIV-1/HIV-2 PLUS O EIA was
estimated from the results of testing 313 patients with AIDS. A
positive test result was obtained for 313 of 313 patients for an
estimated sensitivity in this study of 100% (95% confidence
interval: 99.84% to 100%).
42
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
E
U
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Reactivity in Specimens from High-Risk Individuals from the
United States and Canada
A total of 1011 specimens from high-risk individuals from public
health labs in the United States and Canada were tested with the
GS HIV-1/HIV-2 PLUS O EIA. Results of testing individuals from
the United States (n = 761) and Canada (n = 250) are shown in
Table 9. All specimens were screened with one or more FDA
and/or Canadian licensed HIV-1/HIV-2 EIAs. All specimens
repeatedly reactive with the GS HIV-1/HIV-2 PLUS O EIA and/or
the licensed HIV-1/HIV-2 EIAs were tested with a licensed HIV-1
Western blot. If a specimen tested negative or indeterminate on
the licensed HIV-1 Western blot, it was tested with a licensed
HIV-2 EIA. If a specimen was repeatedly reactive on the licensed
HIV-2 EIA, and negative or indeterminate on the HIV-1 Western
blot, it was additionally tested with an in-house HIV-2 Western
blot.
EN
C
Table 9: Reactivity in Specimens from High-Risk Individuals from the United
States and Canada
No. Tested
GS HIV-1/HIV-2 PLUS O
EIA Repeatedly Reactive
No. RR on one or more
licensed
HIV-1/HIV-2 EIA
No. Pos. by HIV-1
Western blot
U.S
761
36 (4.7%)a
22 (2.9%)a
17 (2.3%) a
Canada
250
1011
3 (1.2%)b
39 (3.9%)
2 (0.8%)b
24 (2.4%)
2 (0.8%) b
19 (1.9%)
EF
R
Total
ER
Group
FO
R
a. Seventeen (17) specimens were repeatedly reactive on both the GS HIV-1/HIV-2 PLUS O EIA and one or more
licensed HIV-1/HIV-2 EIAs.
b. Two (2) specimens were repeatedly reactive on both the GS HIV-1/HIV-2 PLUS O EIA and one or more
licensed HIV-1/HIV-2 EIAs.
Twenty-five specimens were additionally tested with a licensed
HIV-2 EIA (20 specimens were repeatedly reactive on the
GS HIV-1/HIV-2 PLUS O EIA only, and 5 specimens were
repeatedly reactive on the licensed HIV-1/HIV-2 EIA.) All 25
specimens were negative or indeterminate on a licensed HIV-1
Western blot. Of the 25 specimens tested with a licensed HIV-2
EIA, none were repeatedly reactive.
Therefore, the GS HIV-1/HIV-2 PLUS O EIA detected all HIV-1
confirmed positives [19/19 (100%)] in this study of high-risk
populations in the United States and Canada.
43
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
EN
C
E
U
SE
O
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Reactivity in Prospectively Obtained Public Health
Specimens
The GS HIV-1/HIV-2 PLUS O EIA was evaluated in prospective
public health populations. The samples collected and tested at
two public health labs excluded individuals reporting high-risk
behaviors, whereas the third lab did not exclude such high-risk
individuals. The data include 1501 serum specimens tested at
two U.S. and one Canadian location. All specimens were tested
with the GS HIV-1/HIV-2 PLUS O EIA and an FDA licensed
HIV-1/HIV-2 EIA. Specimens repeatedly reactive with the GS
HIV-1/HIV-2 PLUS O EIA and/or the licensed HIV-1/HIV-2 EIA
were tested with a licensed HIV-1 Western blot. Specimens that
were repeatedly reactive with the GS HIV-1/HIV-2 PLUS O EIA
and/or the licensed HIV-1/HIV-2 EIAs were tested with a licensed
HIV-2 EIA if the HIV-1 Western blot was negative or
indeterminate. If a specimen was repeatedly reactive on the
licensed HIV-2 EIA, and negative or indeterminate on the HIV-1
Western blot, it was tested with an in-house HIV-2 Western blot.
ER
Table 10: Reactivity with GS HIV-1/HIV-2 PLUS O EIA Prospective Public
Health Specimens
Repeatedly Reactive
Specimens
Group
NonInitially Repeatedly
Reactive Reactive
Reactive
Licensed EIA
Repeatedly
Reactive
HIV-2 EIA Pos. by HIV-1
Repeatedly Immunoblot
Reactive
alone
Site 1
FO
R
Number
Tested
R
EF
Results Obtained with
GS HIV-1/HIV-2 PLUS O EIA
501
495
(100.00%) (98.80%)
6
(1.20%)
2
(0.40%)
1
(0.20%)
1*
0
Site 2
500
481
(100.00%) (96.20%)
19
(3.80%)
18
(3.60%)
17
(3.40%)
0
13**
Site 3
500
498
(100.00%) (99.60%)
2
(0.40%)
2
(0.40%)
3
(0.60%)
0
1**
SUBTOTAL:
1501
1474
(100.00%) (98.20%)
27
(1.80%)
22
(1.47%)
21
(1.40%)
1
14**
(Minus HIV-1
Immunoblot
Positive)
TOTAL:
1501
- 14
1487
1474
(100.00%) (99.13%)
27
-14
13
(0.87%)
22
-14
8
(0.54%)
21
-14
7
(0.47%)
*This specimen, which was repeatedly reactive only on the GS HIV-1/HIV-2 PLUS O EIA, was indeterminate
when tested on an in-house HIV-2 Western Blot.
**These specimens were repeatedly reactive on both the GS HIV-1/HIV-2 PLUS O EIA and the licensed
HIV-1/HIV-2 EIA.
44
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
SE
O
N
LY
Of the 1501 specimens tested, 14 were repeatedly reactive on
both the GS HIV-1/HIV-2 PLUS O EIA and the licensed HIV-1
EIA, and confirmed as HIV-1 positive on a licensed Western Blot.
As shown in Table 10, of the remaining 1487 prospective public
health specimens not confirmed as positive for HIV-1 or HIV-2, 8
(0.54%) specimens were repeatedly reactive with the GS
HIV-1/HIV-2 PLUS O EIA. In this sample population, seven
(0.47%) specimens were repeatedly reactive with the licensed
HIV-1/HIV-2 EIA. These 15 specimens (8 repeatedly reactive with
the GS HIV-1/HIV-2 PLUS O EIA and 7 repeatedly reactive with
the licensed HIV-1/HIV-2 EIA) were negative or indeterminate on
a licensed HIV-1 Western blot and therefore tested with a
licensed HIV-2 EIA. Of the 15 specimens tested with a licensed
HIV-2 EIA, 1 was repeatedly reactive. This specimen was
indeterminate when tested with an in-house HIV-2 Western blot.
EN
C
E
U
Therefore, the GS HIV-1/HIV-2 PLUS O EIA detected all HIV-1
confirmed positives [14/14 (100%)] in this study of prospective
public health populations in the U.S. and Canada.
FO
R
R
EF
ER
Reactivity with HIV-1 Commercial Seroconversion Panels
The GS HIV-1/HIV-2 PLUS O EIA was tested with specimens
from 50 commercially available seroconversion panels and
compared to two FDA licensed HIV-1/HIV-2 EIAs and licensed
HIV-1 Western blots. As shown in Table 11, the GS HIV-1/HIV-2
PLUS O EIA was equivalent [12/46* (26%)] or more sensitive
[34/46* (74%)] when results were compared to one of the FDA
licensed HIV-1/HIV-2 EIAs. The GS HIV-1/HIV-2 PLUS O EIA was
equivalent [35/50 (70%)], more sensitive [9/50 (18%)], or less
sensitive [6/50 (12%)] when results were compared to a second
FDA licensed HIV-1/HIV-2 EIA. The GS HIV-1/HIV-2 PLUS O EIA
was equivalent [13/50 (26%)] or more sensitive [37/50 (74%)]
when compared to licensed HIV-1 Western blot results.
Therefore, the GS HIV-1/HIV-2 PLUS O EIA was equivalent or
better than licensed HIV-1/HIV-2 EIA tests and licensed HIV-1
Western blots for detection of antibody in HIV-1 seroconversion
samples.
45
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Table 11: Reactivity with 50 HIV-1 Commercial Seroconversion Panels
HIV-1/HIV-2 PLUS O
Equivalent
HIV-1/HIV-2 PLUS O
More Sensitive
HIV-1/HIV-2 PLUS O
Less Sensitive
vs. Licensed Kit #1
12/46*
(26%)
34/46*
(74%)
0
(0%)
vs. Licensed Kit #2
35/50
(70%)
9/50
(18%)
6/50
(12%)
vs. Licensed Western Blot
13/50
(26%)
37/50
(74%)
0
(0%)
*Four of the 50 seroconversion panels did not have test results with the licensed HIV-1/HIV-2 EIA Kit #1 and
are no longer available for testing.
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U
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O
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Reactivity with BBI Panels
When tested with four BBI Panels (Mixed Titer PRB203, Low
Titer PRB105, African HIV Series AfrRB1, and World Wide
WWRB301), 130/130 HIV positive members (100.0%) were
reactive with the GS HIV-1/HIV-2 PLUS O EIA; 124/130 HIV
positive members (95.4%) were reactive with an FDA licensed
HIV-1/HIV-2 EIA; 128/130 HIV positive members (98.5%) were
reactive with a second FDA licensed HIV-1/HIV-2 EIA.
FO
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ER
EN
C
Reactivity in Preselected Specimens from Individuals
Positive for HIV-2 Antibodies and Confirmed by Western blot
A total of 302 specimens, obtained from HIV-2 confirmed
antibody positive individuals, were tested with the GS
HIV-1/HIV-2 PLUS O EIA. All specimens were repeatedly reactive
with a licensed HIV-2 EIA and positive on an in-house HIV-2
Western blot. All of the 302 specimens tested were classified as
repeatedly reactive with the GS HIV-1/HIV-2 PLUS O EIA for an
estimated sensitivity in this study of 100% (95% confidence
interval: 99.83% – 100%).
Reactivity in Specimens Known to be Positive for Antibodies
to HIV-1 Group O
Seventy-seven different specimens known to be positive for
antibodies to HIV-1 Group O (characterized by serotype and/or
genotype) were tested with the GS HIV-1/HIV-2 PLUS O EIA. All
[77/77 (100%)] of the HIV-1 Group O positive samples tested
with the GS HIV-1/HIV-2 PLUS O EIA were initially or repeatedly
reactive. (One sample was initially reactive and had insufficient
volume for repeat testing.) The HIV-1 Group O sensitivity of the
46
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
GS HIV-1/HIV-2 PLUS O EIA in this study was 100%, with a 95%
confidence interval of 99.35% to 100%.
Reactivity of Known Positives using the EVOLIS® Automated
Microplate System
Comparative studies were performed with specimens known to
be positive for HIV-1, HIV-2, and HIV-O antibody, including
dilution series and seroconversion panels, using the EVOLIS®
Automated Microplate System in comparison to manual testing.
EN
C
E
U
SE
O
N
LY
A total of 100 known positive HIV-1 samples were tested once
on each of three different instruments at each of three different
sites. Approximately 60-80% of the known positive HIV-1
samples were diluted to be near the assay cut-off. For the 100
known positive HIV-1 samples combined across sites, 97.3%
(292/300) of manual results were reactive; 100% (300/300) of the
EVOLIS® Automated Microplate System results were reactive.
The positive % agreement was 100% (292/292) with a 95%
confidence interval range of 98.7%-100.0%.
FO
R
R
EF
ER
A total of 100 known positive HIV-2 samples were tested once
on each of three different instruments at each of three different
sites. Approximately 60-80% of the known positive HIV-2
samples were near the assay cut-off. For the 100 known positive
HIV-2 samples averaged across sites, 100% (100/100) of manual
results were reactive; 100% (100/100) of the EVOLIS®
Automated Microplate System results were reactive. The
positive % agreement was 100% (100/100) with a 95%
confidence interval range of 100.0%-100.0%.
The mean quantitative S/CO for the HIV-2 test panel was
10.95% lower on EVOLIS® as compared to manual testing. The
difference was statistically significant, but not clinically
significant.
These studies demonstrate comparable results for both methods
when testing for HIV-1, HIV-2, and HIV-1 Group O antibodies
with the GS HIV-1/HIV-2 PLUS O EIA.
47
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
Reactivity of Known Positives using the Elite™ Automated
Microplate System
Comparative studies were performed with specimens known to
be positive for HIV-1 group M, HIV-2, and HIV-1 group O
antibody, including dilution series and seroconversion panels,
using the Elite™ Automated Microplate System in comparison to
manual testing.
U
SE
O
N
LY
A total of 100 known HIV-1 group M positive samples were
tested once on each of three different instruments at each of
three different sites. Approximately 60-80% of the known HIV-1
positive samples were diluted to be near the assay cut-off. All of
these positive samples were reactive at all 3 sites, with both the
Elite™ Automated Microplate System and manual testing.
Averaged across all sites, the positive % agreement was 100%
(100/100).
FO
R
R
EF
ER
EN
C
E
Performance of the Elite™ Automated Microplate System and
manual testing were compared with 100 known HIV-2 positive
samples that were tested once on each of three different
instruments at each of three different sites. Approximately 6080% of the known HIV-2 positive samples were near the assay
cut-off. For the 100 known HIV-2 positive samples averaged
across sites, 97% (97/100) of manual results were reactive and
100% (100/100) of the Elite™ Automated Microplate System
results were reactive. The overall % agreement, averaged
across all sites, was 97% (97/100).
A total of 50 known HIV-1 group O positive samples were tested
once on each of three different instruments at each of three
different sites. Approximately 40-60% of the known HIV-1
positive samples were low positives. Averaged across all 3 sites,
98% (49/50) of the Elite™ Automated Microplate System and
98% (49/50) of the manual testing results were reactive. The
overall % agreement, averaged across all sites, was 100%
(50/50).
48
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
These studies demonstrate comparable results for both methods
when testing for HIV-1, HIV-2, and HIV-1 Group O antibodies
with the GS HIV-1/HIV-2 PLUS O EIA.
EN
C
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U
SE
O
N
LY
Reactivity of Known Positives Using the ORTHO® Summit
System
Head-to-head studies with dilution series, seroconversion series,
and specimens known to be positive for HIV-1, HIV-2, and HIV-1
Group O antibody were performed using the ORTHO® Summit
System with the ORTHO® Summit Sample Handling System* and
the Bio-Rad manual equipment method of testing. Studies were
also performed using the ORTHO® Summit Sample Handling
System in comparison to the ORTHO VERSEIA® Pipetter. From
these comparative studies, it was concluded that the GS
HIV-1/HIV-2 PLUS O EIA assay results are acceptable for HIV-1,
HIV-2, and HIV-1 Group O analytes using manual testing or
using either of these ORTHO® pipetters with the ORTHO®
Summit System.
FO
R
R
EF
ER
Additional studies were performed with the stand-alone ORTHO
VERSEIA® Pipetter and a stand-alone ORTHO® Summit
Processor (OSP) in comparison with the ORTHO VERSEIA®
Integrated Processor (VIP). These studies used dilution panels
and known positive samples. With the HIV-1 dilution panels, the
VIP showed an overall S/CO percent difference increase from
the stand-alone system of 7.6%, and with the HIV-2 panels, the
VIP showed an overall S/CO percent difference increase of
8.8%. For the HIV-1 known positive samples, the VIP showed a
percent difference increase in S/CO of 7.1%, and for the HIV-2
known positive samples, the VIP showed a percent difference
increase of 10.9%.
*
The ORTHO® Summit Sample Handling System is now a legacy device no longer
available for marketing.
49
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
14-PERFORMANCE CHARACTERISTICS OF
CADAVERIC SPECIMEN TESTING
O
N
LY
REPRODUCIBILITY
Inter-assay reproducibility of the GS HIV-1/HIV-2 PLUS O EIA
was assessed using nineteen post-mortem sera and twenty
normal donor sera, spiked with HIV-1 and HIV-2 positive serum
to give reactivity near the cutoff. Each of the samples was tested
once on three different days on each of three lots of the GS
HIV-1/HIV-2 PLUS O EIA at one site. For inter-assay
reproducibility over all lots, percent coefficient of variation (%CV)
ranged from 5.84% to 22.37% for the post mortem samples, and
from 4.84% to 21.44% for the normal donor samples.
EF
ER
EN
C
E
U
SE
SPECIFICITY
Specificity was evaluated in a clinical investigation at one site in
three studies. In total, ninety-five (95) post-mortem samples and
ninety-five (95) normal donor samples were tested concurrently
on five lots of the GS HIV-1/HIV-2 PLUS O EIA. Repeatedly
reactive specimens were additionally tested with a licensed
HIV-1/HIV-2 EIA and confirmed with a licensed HIV-1 Western
blot. Results are presented in Table 12 below.
Post-mortem
Normal Donor
Number
Tested
Nonreactive
95
95
R
Population
FO
R
Table 12: Reactivity with GS HIV-1/HIV-2 PLUS O EIA
Initially
Reactive
Repeatedly
Reactive
Confirmed
Positive
95 (100.0%)
0 (0.00%)
NA
NA
94 (98.95%)
1* (1.05%)
1*
0
NA = Not Applicable
*This specimen was nonreactive by a licensed HIV-1/HIV-2 EIA and negative by HIV-1 Western Blot.
Specificity of the GS HIV-1/HIV-2 PLUS O EIA in testing
cadaveric specimens was estimated by the following formula:
(# specimens - # repeatedly reactive specimens)
(# specimens - # repeatedly reactive specimens confirmed
positive for HIV-1 or HIV-2)
50
x100
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
N
LY
A total of ninety-five (95) unselected post-mortem specimens
were tested with the GS HIV-1/HIV-2 PLUS O EIA for determining
specificity. All post-mortem specimens were compared to
normal donor specimens. None of the post-mortem specimens
were reactive with the GS HIV-1/HIV-2 PLUS O EIA. Thus, the
GS HIV-1/HIV-2 PLUS O EIA has an estimated specificity of
100% (95% binomial confidence interval = [99.47%, 100%]). By
comparison, one of the ninety-five normal donor specimens
tested concurrently (1.05%) was initially and repeatedly reactive,
but did not confirm positive for HIV-1 or HIV-2. The mean optical
density for the 95 post-mortem samples was 0.054, whereas the
mean for the 94 nonreactive normal donor samples was 0.044.
FO
R
R
EF
ER
EN
C
E
U
SE
O
SENSITIVITY
Ninety-five (95) post-mortem samples and ninety-four (94)
normal donor samples were pre-screened for antibody to HIV-1
and HIV-2 and found to be nonreactive. Each sample was
divided into two portions. One portion of each post-mortem and
normal donor sample was spiked at a potency near cutoff with a
positive serum containing HIV-1 or HIV-2 antibody, and the
remaining portion was left unspiked. The ninety-five spiked and
unspiked post-mortem samples were tested concurrently with
ninety-four spiked and unspiked normal donor specimens on the
same run of the GS HIV-1/HIV-2 PLUS O EIA. Spiked specimens
were expected to be reactive and therefore were not retested in
duplicate. Results are presented in Table 13 below.
Table 13: Reactivity with GS HIV-1/HIV-2 PLUS O EIA
Number
Tested Nonreactive
Population
Initially
Reactive
Repeatedly
Reactive
Confirmed
Positive
Spiked Post-mortem
Unspiked Post-mortem
95
95
0 (0.00%) 95 (100.0%)
95 (100.0%) 0 (0.00%)
NT
NA
95 (100.0%)
Spiked Normal Donor
Unspiked Normal Donor
94
94
0 (0.00%) 94 (100.0%)
94 (100.0%) 0 (0.00%)
NT
NA
94 (100.0%)
NT = Not Tested
NA
NA
NA = Not Applicable
As can be seen in the table above, of ninety-five post-mortem
samples and ninety-four normal donor samples, spiked at a
potency near cutoff and tested concurrently, all (100.00%) were
reactive with the GS HIV-1/HIV-2 PLUS O EIA (95% binomial
51
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confidence interval = [99.47%, 100%]). These results
demonstrate that the detection of HIV-1 and HIV-2 antibody in
post-mortem samples is comparable to the detection in normal
donors.
52
FOR REFERENCE USE ONLY: DO NOT USE in place of package inserts provided with each test kit.
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For Customer Orders or Technical Service call:
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